Analysis of Five Arboviruses and Culicoides Distribution on Cattle Farms in Jeollabuk-Do, Korea

ISSN (Print) 0023-4001 ISSN (Online) 1738-0006

Korean J Parasitol Vol. 56, No. 5: 477-485, October 2018 ▣ ORIGINAL ARTICLE https://doi.org/10.3347/kjp.2018.56.5.477

Analysis of Five Arboviruses and Culicoides Distribution on Cattle Farms in Jeollabuk-do, Korea

Daram Yang1, Myeon-Sik Yang1, Haerin Rhim1, Jae-Ik Han1, Jae-Ku Oem1, Yeon-Hee Kim2, Kyoung-Ki Lee2, 1 1, Chae-Woong Lim , Bumseok Kim * 1College of Veterinary Medicine and Korea Zoonosis Research Institute, Chonbuk National University, Iksan 54596, Korea; 2Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea

Abstract: Arthropod-borne viruses (Arboviruses) are transmitted by arthropods such as Culicoides biting midges and cause abortion, stillbirth, and congenital malformation in ruminants, apparently leading to economic losses to farmers. To monitor the distribution of Culicoides and to determine their relationship with different environmental conditions (temperature, humidity, wind speed, and altitude of the farms) on 5 cattle farms, Culicoides were collected during summer season (May-September) in 2016 and 2017, and analyzed for identification of species and detection of arboviruses. About 35% of the Culicoides were collected in July and the collection rate increased with increase in temperature and humidity. The higher altitude where the farms were located, the more Culicoides were collected on inside than outside. In antigen test of Culicoides against 5 arboviruses, only Chuzan virus (CHUV) (2.63%) was detected in 2016. The Akabane virus (AKAV), CHUV, Ibaraki virus and Bovine ephemeral fever virus (BEFV) had a positive rate of less than 1.8% in 2017. In antigen test of bovine whole blood, AKAV (12.96%) and BEFV (0.96%) were positive in only one of the farms. As a result of serum neutralization test, antibodies against AKAV were generally measured in all the farms. These results suggest that vaccina- tion before the season in which the Culicoides are active is probably best to prevent arbovirus infections.

Key words: Culicoides biting midges, arbovirus, akabane virus, cattle, Korea

INTRODUCTION in the veterinary field and are transmitted by arthropods such as Culicoides biting midges [1]. Arboviruses infected with rumi- Culicoides biting midges (Diptera: Ceratopogonidae) are the nants cause abortion, stillbirth, and congenital malformation smallest haematophagous insects, which transmit the fatal vi- [6,7]. Akabane virus (AKAV) and Aino virus (AINOV) belong rus to ruminants [1]. More than 1,400 species of Culicoides to the family Peribunyaviridae [1,8]. Sporadic outbreaks of AKAV have been described and present on all land with the excep- have been reported widely in Japan, Southeast Asia and the tion of Antarctica and New Zealand [1,2]. A size of Culicoides Middle East, including Korea [9-12]. In Japan, JaGAr39, the is around 1-3 mm, and they have a complete metamorphosis prototype of AKAV, was first isolated in 1959 [9]. In Korea, a life cycle going through the egg, larva, pupa, and adult stages case of AKAV was first reported in 1980 [12]. In 2010, meningi- [1,3]. Females preparing to spawn after mating with males tis associated with AKAV infection occurred in a large-scale in suck up blood of human or animal to get animal protein at South Korea [13]. More than 500 cattle exhibited symptoms dawn or dusk [1,3]. Breeding sites of Culicoides are watery and such as movement disorder, trembling, and in severe cases, the nutritious streams that can help them grow and develop larvae cattle exhibited an inability to stand. Chuzan virus (CHUV) [4]. Adult Culicoides have different wing patterns based on spe- and Ibaraki virus (IBAV) are members of the family Reoviridae cies, which helps in easy identification of different species [5]. [1,14]. CHUV was first emerged in Japan, 1985, and the clini- Arthropod-borne viruses (arboviruses) are major pathogens cal signs are neurological abnormalities and hydranencephaly [15]. IBAV infection is characterized by fever, salivation, anorex-

Received 13 March 2018, revised 16 August 2018, accepted 2 September 2018. ia, and a deglutitive disorder in cattle. Symptoms except deglu- • Corresponding author ([email protected]) * titive disorder with dysphagia are usually mild. Bovine ephem- © 2018, Korean Society for Parasitology and Tropical Medicine eral fever virus (BEFV) grouped in the family Rhabdoviridae is This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) similar to the IBAV, which is characterized by acute fever [14]. which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. High fever, nasal discharges, salivation, and inability to stand

477 478 Korean J Parasitol Vol. 56, No. 5: 477-485, October 2018

appear to the principal signs of cattle infected with BEFV. collection date and species. The surface temperature of the earth is rising continuously During June and July, whole blood of cattle (over six months) due to global warming, which is expected to rise further [3]. was drawn from the jugular vein using 10 ml syringes. Collect- Temperature is one of the environmental factors that affect the ed samples were into vacuum tubes treated anticoagulant, eth- activity of poikilothermic Culicoides [16,17]. Viruses that spread ylenediaminetetraacetic acid (EDTA), and mixed. faster due to increased vector activity in warmer atmospheric temperatures result in severe economic losses in the industrial Measurement of temperature, humidity, and wind speed animal field. The aim of this study was to investigate the Culi- The temperature and humidity were measured using a digi- coides distribution in different cattle farms in the area where en- tal thermo-hygrometer machine (LAVISEN, Namyangju, Ko- cephalomyelitis occurred on a large-scale in 2010, and environ- rea) and the wind speed data were obtained through the Korea mental conditions which affect the Culicoides abundance. Meteorological Administration (http://www.kma.go.kr).

MATERIALS AND METHODS DNA/RNA extraction and Reverse transcription PCR analysis Collection and identification of Culicoides biting midges Pooled Culicoides samples were homogenized using a Hand- Culicoides biting midges were collected once (2016) or twice held Pestle Cordless Motor (BIOFACT, Daejeon, Korea) with (2017) per month using a light trap (SNC, Hanam, Korea) 500 μl of Dulbecco’s Modified Eagle’s Medium (DMEM). Ho- from 5 different cattle farms located in Gimje (35˚50’34.4389’’ mogenized samples were centrifuged at 13,000 rpm for 10 N; 126˚51’21.3368’’ E), Gochang (35˚24’42.0937’’ N; 126˚39’05. min at 4˚C and the supernatant was used for DNA/RNA extrac- 0940’’ E), Wanju (36˚03’20.7356’’ N; 127˚11 ’12.1909’’ E), Jinan tion using a Patho Gene-spinTM DNA/RNA Extraction Kit (iN- (35˚51’17.6353’’ N; 127°20’19.2646’’ E) and Namwon (35˚29’32. tRON, Seongnam, Korea) according to the manufacturer's in- 7782’’ N; 127˚38’42.2509’’ E) areas, Jeollabuk Province from structions. Bovine whole blood was centrifuged at 13,000 rpm May to September in 2016 and 2017 (Supplementary Fig. S1). for 5 min to get buffy coat and plasma. Collected supernatant Each region differed in altitudes (about 50 m, 50 m, 300 m, was used for virus DNA/RNA extraction using a MinElute Vi- 500 m and 600 m respectively). rus Spin Kit (QIAamp, Hilden, Germany). Used light trap consisted of 8 W UV fluorescent light with a Extracted Culicoides and blood DNA/RNA samples were test- downdraft suction fan. The traps were located inside and out- ed to detect 5 arboviruses (AKAV, AINOV, CHUV, IBAV and side (about 200 m from breeding farms) the farms to compare BEFV) using VDx® Single RT-PCR Kit (MEDIAN Diagnostics, the differences in collection rates. The traps were set in the af- Chuncheon, Korea). Table 1 showed the information includ- ternoon before sunset and were collected the next morning ing primer sequences used, target gene, and size. PCR program within 1 hr after sunrise. Collected Culicoides samples were is as follows: cDNA synthesis at 50˚C for 30 min and initial in- sorted morphologically into species according to their wing activation at 95˚C for 15 min in the first cycle followed by 40 patterns observed under the microscope [18,19]. After classifi- cycles of denaturation at 94˚C for 20 sec, annealing at 55˚C for cation, Culicoides (-30) were pooled into one sample based on 30 sec and extension at 72˚C for 40 sec. Lastly, a final exten-

Table 1. Sequences of primer used and target information

No. Target Virus Primer Gene Size (bp) 1 AKAV J424F: 5’-CAGAAGAAGGCCAAGATGGT-3’ S segments 476 J425R: 5’-AATGCAGCCTTGACTGCGTCC-3’ N gene 2 AINOV J424F: 5’-CAGAAGAAGGCCAAGATGGT-3’ S segments 584 J426R: 5’-GGGTGGGGTTTTACAGGAA-3’ N gene 3 CHUV J427F: 5’-CTGGCTTTCTGAGGCGTTTC-3’ NS1 (S5) 306 J428R: 5’-GGTTGCTCAATATGCCAAGCGA-3’ 4 IBAV J431F: 5’-AGGATACGGAGGCGGCCTTCTT-3’ S segment3 411 J432R: 5’-CCGGAGATACCTCCATTACC-3’ 5 BEFV J429F: 5’-CGGTTGCACAGATGCGGTTAAG-3’ G 262 J430R: 5’-GACTCTCACATCTGGTATCC-3’ Yang et al.: Analysis of Culicoides biting midges in cattle farms 479

sion was carried out at 72˚C for 5 min in the last cycle [20]. C-1586) were maintained in alpha- minimum essential medium (Gibco, Grand Island, New York, USA) supplemented with Gel electrophoresis 5% fetal bovine serum and antimycoticantibiotics (Gibco). Bo- Amplification samples (5 μl) were analyzed by electropho- vine serum diluted to 2-fold serial was mixed with equal vol- resis in 1.5% agarose gels (GeneDireX®, Taiwan) containing umes of virus containing 200 TCID50/0.1 ml and inoculated 0.5 ml of RedSafeTM (iNtRON). Typically, 6 μl of 100 bp mark- with Vero cells [8,20,21]. The plates were microscopically ex- er and samples were loaded in the gel and run at 100 V for 25 amined after 3 and 5 days to find virus-specific cytopathic ef- min. Subsequently, the gel images were captured using Im- fects (CPE). Antibody titer was defined as the reciprocal of the ageQuantTM LAS 500 (GE Healthcare Life Science, Pittsburgh, highest serum dilution at which CPE was inhibited. A titer of 4 Pennsylvania, USA). folds or greater was considered to be positive.

Serology analysis in bovine blood against arboviruses RESULTS The AKAV strain 93FMX (KVCC-VR63), AINOV strain KSA 9910 (Korea Veterinary Culture Collection (KVCC) VR64), Collected numbers of Culicoides species CHUV strain YongAm (KVCC-VR66), IBAV strain 08220 (KVC- In 2016, a total of 4,932 Culicoides were collected (Table 2; CVR65) and BEFV strain TongRae (KVCC VR41) were used for Fig. 1A). Collected Culicoides species were C. arakawae (n =2,988, serum neutralization tests (SNT) [8,20]. Vero cells (ATCC, 60.6%), C. punctatus (n=1,497, 30.4%), C. nipponensis (n=246,

Table 2. Information about collected Culicoides species in 2016 and 2017

Species Year Region Total number C. arakawae C. punctatus C. tainanus C. nipponensis Culicoides spp. 2016 Wanju 605 272 (45.0) 264 (43.5) 36 (6.0) 0 33 (5.5) Gochang 1,237 868 (70.2) 93 (7.4) 2 (0.2) 246 (19.9) 28 (2.3) Gimje 304 295 (97.0) 6 (2.0) 2 (0.7) 0 1 (0.3) Jinan 1,328 996 (75.0) 294 (22.1) 9 (0.7) 0 29 (2.2) Namwon 1,458 557 (38.2) 840 (57.6) 12 (0.8) 0 49 (3.4) Total 4,932 2,988 (60.6) 1,497 (30.4) 61 (1.2) 246 (5.0) 140 (2.8) 2017 Wanju 664 348 (52.4) 309 (46.5) 4 (0.6) 0 3 (0.5) Gochang 2,904 2,636 (90.8) 165 (5.7) 2 (0.1) 87 (3.0) 14 (0.5) Gimje 220 215 (97.7) 5 (2.3) 0 0 0 Jinan 2,981 2,129 (71.4) 813 (27.3) 23 (0.8) 0 16 (0.5) Namwon 3,954 2,011 (50.9) 1,927 (48.7) 6 (0.2) 0 10 (0.3) Total 10,723 7,339 (68.4) 3,219 (30.0) 35 (0.3) 87 (0.8) 43 (0.4)

2016 2017 A 1,200 B 3,000 Wanju Wanju 1,000 Gochang 2,500 Gochang Gimje Gimje 800 Jinan 2,000 Jinan Namwon Namwon 600 1,500

400 1,000

Total collected number Total 200 collected number Total 500

0 0 C. arakawae C. punctatus C. tainanus C. nipponensis Culicoides spp. C. arakawae C. punctatus C. tainanus C. nipponensis Culicoides spp.

Fig. 1. Collected Culicoides species and numbers in 2016 (A) and 2017 (B). According to results of Culicoides collection in 2016 (once per month) (A) and 2017 (twice per month) (B), dominant species was C. arakawae, followed by C. punctatus. C. nipponensis was only collected in Gochang. 480 Korean J Parasitol Vol. 56, No. 5: 477-485, October 2018

5.0%), C. tainanus (n=61, 1.2%) and Culicoides spp. (n=140, The outside of the cattle farms located at low altitude (about 2.8%). Dominant species were C. arakawae in 4 farms whereas 50 m) showed higher collection rates than inside the farm (Fig. C. punctatus was dominant in one farm (Namwon). C. nippon- 2). On the other hand, collection rates of Culicoides were high- ensis was collected only in one farm (Gochang). er inside the farm located at high altitude (over 300 m) com- A total of 10,723 Culicoides were collected in 2017 (Table 2; pared to the outside of cattle farms. Fig. 1B). Species such as C. arakawae (n=7,339, 68.4%), C. punctatus (n=3,219, 30.0%), C. nipponensis (n=87, 0.8%), C. Distribution of collected Culicoides species month-wise tainanus (n=35, 0.3%) and Culicoides spp. (n=43, 0.4%) were Overall, the number of collected Culicoides was mainly concen- collected. C. arakawae was dominant species in all the regions trated in July 2016 (Fig. 3A) and 2017 (Fig. 3B). A total of 1,871 and C. nipponensis was collected only in one farm (Gochang) Culicoides were collected in July followed by June (n=1,084), Au- similar to the collection in 2016. gust (n=967), September (n=740), and May (n=307) in 2016. In 2017, a total of 3,618 Culicoides were collected in July and fol- Comparison of collected Culicoides numbers between lowed by August (n=3,075), June (n=2,063), May (n=1,002), inside and outside the cattle farms and September (n=965). Generally, C. arakawae was the most Different collection rates of Culicoides inside and outside the collected species during each month. cattle farms were observed depending on different altitudes. Correlation between collection rates of Culicoides and 6,000 temperature, humidity, and wind speed

5,000 Three environmental factors (temperature, humidity, and wind speed) were recorded on the collection dates of Culicoides. 4,000 The mean temperature was in the range of 16.4-26.9˚C, and the 3,000 mean humidity was in the range of 67.4-88.1%. The mean wind 2,000 speed ranged from 1 to 2 m/sec. On the conditions of 25.3˚C

Total No. of Culicoides Total temperature, 87.3% humidity, and 1.6 m/sec of mean wind 1,000 speed, the highest mean number of Culicoides collected was 364. 0 Wanju Gochang Gimje Jinan Namwon On the other hand, the lowest mean number of Culicoides col- 300 m 50 m 50 m 500 m 600 m lected was 91 at 20.2˚C temperature, 80% humidity, and 1.8 m/ sec of mean wind speed. Culicoides tended to be more collected Fig. 2. Comparison of numbers of Culicoides collected between inside and outside the cattle farms. A majority of Culicoides were at a high mean temperature and humidity (Fig. 4A, B). There collected from low altitude area outside the farms compared to was no effect of mean wind speed on the rates of Culicoides col- inside the farm. At farms located at relatively high altitude, more lection (Fig. 4C). Culicoides were collected from inside the farm as compared to outside the farm.

A 2016 B 2017 2,000 4,000 * C. arakawae C. arakawae C. punctatus * C. punctatus 1,600 C. tainanus C. tainanus C. nipponensis 3,000 C. nipponensis * Culicoides spp. 1,200 Culicoides spp. * * * 2,000 800 *

Total No. of Culicoides Total 1,000 400 * * * No. of Culicoides Total 0 0 May Jun Jul Aug Sep May Jun Jul Aug Sep

Fig. 3. Distribution of collected Culicoides species in the months of 2016 (A) and 2017 (B). *Total number of collected culicoicles species. Yang et al.: Analysis of Culicoides biting midges in cattle farms 481

Antigen detection of Culicoides and bovine whole blood located in Jinan with a positive rate of 1.81%. Two CHUV were against 5 arboviruses detected in one C. arakawae pooling and one C. punctatus Collected Culicoides were conducted to test the positive rates pooling at the farm located in Namwon with a positive rate of against 5 arboviruses. In 2016, only CHUV was detected twice 1.20%. IBAV and BEFV were positive in each C. punctatus and in Jinan cattle farm with a positive rate of 2.63% in 2 of the 76 C. arakawae at the farm located in Wanju with a positive rate poolings (Table 3A). Each species of poolings showing posi- of 0.60%. In antigen test of bovine blood against 5 arbovirus- tive were C. arakawae and C. punctatus. In 2017, 3 of the 166 es, the antigen to AKAV (12.96%) and antigen to BEFV poolings were positive for AKAV, 2 for CHUV, one for IBAV, (0.93%) were detected in samples collected from the farm lo- and one for BEFV. AKAV were detected in one C. arakawae cated in Gochang (Table 3B). To confirm the identification of pooling at Gochang and 2 C. arakawae poolings at the farm arbovirus-specific detection, Basic Local Alignment Search

400 A 364 359.6 350 324.4 300 269.6 290.8 250

200 143 150 102 102.6 91 Average of collected number Average 100 97.8 50 16 18 20 22 24 26 28 Temperature (˚C)

400 B 364 350 324.2 359.6 300 290.8 250 269.6 200 143 150 102.6 97.8 102 91 100 Average of collected number Average 50 65 70 75 80 85 90 Humidity (%) C 450 400 359.6 364 350 290.8 300

250 207.6 200 183.7

150 102.6

Average of collected number Average 100 122.5 50 1.0 1.2 1.4 1.6 1.8 2.0 Wind speed (m/sec)

Fig. 4. Correlation between the number of collected Culicoides and temperature (A), humidity (B), and wind speed (C). More Culicoides were collected at higher temperature and humidity. Collected Culicoides was concentrated at 1.4-1.6 m/s. 482 Korean J Parasitol Vol. 56, No. 5: 477-485, October 2018

Table 3. Antigen detection of Culicoides and bovine whole blood against 5 arboviruses (A) In results of antigen detection rates against arboviruses in Culicoides, AINOV was not detected in any year, and CHUV was detected in 2016 (2.63%) and 2017 (1.20%). All of the detected viruses showed a detection rate within 3% Arboviruses Year AKAV AINOV CHUV IBAV BEFV 2016 - - 2.63% (2/76) - - In Jinan, 1 C. arakawae 1 C. punctatus 2017 1.81% (3/166) - 1.20% (2/166) 0.60% (1/166) 0.60% (1/166) In Gochang, 1 C. arakawae In Namwon, 1 C. arakawae In Wanju, 1 C. punctatus In Wanju, 1 C. arakawae In Jinan, 2 C. arakawae 1 C. punctatus

(B) In bovine blood, only AKAV and BEFV were detected. The test was not carried out in Jinan Arboviruses Region AKAV (%) AINOV (%) CHUV (%) IBAV (%) BEFV (%) Wanju (n= 20) 0 0 0 0 0 Gochang (n= 108) 12.96 (14/108) 0 0 0 0.93 (1/108) Gimje (n= 20) 0 0 0 0 0 Namwon (n= 42) 0 0 0 0 0 Jinan The test was not carried out.

Tool (BLAST) was conducted with bi-directional sequencing sions, such as collection means using UV light with a wide data of positive samples. The results showed that over 92% range of wavelengths [24], and traps in which carbon dioxide identity was observed in individual arboviruses (Supplemen- (CO2) is released at diverse concentrations to attract Culicoides tary Table S1). [25]. They showed that the standard CDC UV light trap collected the Culicoides significantly more than the normal LED

Serum neutralization tests against 5 arboviruses trap, and CO2 trap releasing decanal or phenol collected high-

As a result of antibody test against arboviruses in bovine se- ly compared to trap with CO2 alone. These results will be a rum, antibody to AKAV (3.57%, 25.00%, 5.56%, 33.33%, and useful source to collect Culicoides more effectively. In addition, 11.36%) was positive in all the 5 farms (Table 4A). Antibody new species of Culicoides as a vector of arboviruses are continu- to CHUV (3.57% and 5.00%) was detected in blood samples ously being discovered [25-27]. In Korea, new Culicoides spe- collected from 2 farms (Wanju and Gochang). The antibody cies were discovered in 2013 [28]. Therefore, epidemiological titer of BEFV detected was particularly high (64.29%) in blood studies on arthropod-borne are expected to experience contin- samples collected from the farm located in Wanju (Table 4B). ual growth. There were cases that 2 or more arboviruses were detected in In 2016 and 2017, a total number of 15,655 Culicoides were one cattle in Wanju (21.4%) and Gochang (17.5%). collected during the collection period. The species were classi- fied as C. arakawae, C. punctatus, C. nipponensis and C. tainanus. DISCUSSION In this study, C. arakawae was the extensively collected species while C. arakawae which is a transvector of leucocytozoon dis- Transmission of arthropod-borne viral disease is difficult to ease in Japan was generally collected from the chicken farm prevent as they cannot be visually observed and spread quickly [29]. Previous studies showed that C. arakawae was rarely and widely. Surveillance has steadily been conducted in several found in ruminant farms and is blood-sucking midges which countries to investigate the distribution of Culicoides that is a parasitize chicken [29,30]. There were no chicken farms vector of arboviruses, and to test against target viruses for pre- around the 5 cattle farms where Culicoides were collected in dicting the outbreak [2,22,23]. As these investigations have this study. The detection of antigens to AKAV, CHUV, and been carried out for a long time, haematophagous vector col- BEFV in C. arakawae suggests that C. arakawae can hold arbovi- lection methods have been developed on a variety of occa- ruses antigen although it is not principal vector that transmit Yang et al.: Analysis of Culicoides biting midges in cattle farms 483

Table 4. Serum neutralization tests of bovine blood against 5 arboviruses (A) In data of arboviruses antibody positive based on regions, the AKAV antibody detected in all regions Arboviruses Region AKAV (%) AINOV (%) CHUV (%) IBAV (%) BEFV Wanju 3.57 (1/28) 0 3.57 (1/28) 0 64.29 (18/28) Gochang 25.00 (10/40) 0 5.00 (2/40) 0 5.00 (2/40) Gimje 5.56 (1/18) 0 0 0 5.56 (1/18) Jinan 33.33 (2/6) 0 0 0 0 Namwon 11.36 (5/44) 0 0 0 0

(B) Antibody titer which was measured against arboviruses based on regions was considered to be positive if a titer of 4 folds or greater, and BEF showed high titer Arboviruses positive Arboviruses (2n) Region number of total poolings AKAV AINOV CHUV IBAV BEFV Wanju 18 of 28 <2 <2 <2 <2 32 <2 <2 <2 <2 16 <2 <2 <2 <2 32 <2 <2 <2 <2 16 <2 <2 <2 <2 64 <2 <2 <2 <2 64 4 <2 <2 <2 64 <2 <2 <2 <2 32 2 <2 <2 <2 8 <2 <2 <2 <2 64 <2 <2 <2 <2 32 <2 <2 <2 <2 32 <2 <2 <2 <2 128 2 <2 16 <2 16 2 <2 <2 <2 32 <2 <2 <2 <2 64 2 <2 <2 <2 64 2 <2 <2 <2 16 Gochang 12 of 40 4 <2 <2 <2 <2 4 <2 <2 <2 8 4 <2 2 <2 2 4 2 <2 <2 2 4 <2 <2 <2 <2 4 <2 <2 <2 <2 8 <2 <2 <2 <2 8 <2 4 <2 <2 2 <2 <2 <2 64 4 <2 <2 <2 <2 2 <2 4 <2 <2 4 <2 2 <2 <2 Gimje 2 of 18 8 <2 <2 <2 <2 <2 <2 <2 <2 16 Jinan 2 of 6 8 <2 <2 <2 <2 8 <2 <2 <2 <2 Namwon 5 of 44 4 <2 <2 <2 <2 4 <2 <2 <2 <2 4 <2 <2 <2 <2 4 <2 <2 <2 <2 4 <2 <2 <2 <2 arboviruses to ruminants. did not show any correlation with the type of virus and region. When compared with the antigen detection results against During the study period, there were no reported damages as- arboviruses in Culicoides, the antigen test in the bovine blood sociated with arboviruses, and the appearance and epidemic 484 Korean J Parasitol Vol. 56, No. 5: 477-485, October 2018

of 5 arboviruses seemed to be rare. On the other hand, anti- not easy to conclude the relationship between the number of body test in the bovine serum, antibody to BEFV was detected collected Culicoides and the environmental factors because alti- and the antibody titer was high in one farm (Wanju). As this tude alone exactly affects environmental conditions. Therefore, farm was vaccinated only against AKAV, there is a possibility it is necessary to study whether more various conditions affect that the farm might get infected with BEFV. However, there behavior of Culicoides. Furthermore, through environmental were no virus-related clinical symptoms and the antibody titer studies on farms that can attract or avoid Culicoides, we may can be considered as a maternal antibody. Serological tests us- suggest the manuals that farmers can prevent the occurrence of ing blood were not initially designed and there was a lack of Culicoides related diseases. sample number and collection period. In future studies, a large number of blood samples from various regions will be ACKNOWLEDGMENT employed to perform the more specific serological examina- tion. In this study, because blood samples were obtained from This work was supported by the Co-operative Research Pro- randomly selected cattle, it was difficult to determine the exact gram for Agriculture, Science and Technology Development timing of the virus infection. In order to supplement these (PJ011978072018) in the Rural Development Administration, shortcomings, surveillance study using sentinel cattle that con- Republic of Korea. firmed no-virus infection before investigated year should be conducted. CONFLICT OF INTEREST Previous studies have reported that Culicoides are highly in- fluenced by various environmental factors like temperature, The authors declare that there are no conflicts of interest. humidity, and wind speed [1,31]. 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BLAST results Virus Detected sequence name Identity (%) AKAV F* Akabane virus strain 93 FMX S protein mRNA, partial cds 100 Akabane virus isolate 93FMX nucleocapsid protein (N) gene, complete cds 99 R* Akabane virus strain 93 FMX S protein mRNA, partial cds 99 Akabane virus isolate 93FMX nucleocapsid protein (N) gene, complete cds 98 AINOV F Aino virus N and NSs genes, segment S, genomic RNA, isolate 38K 99 R Aino virus N and NSs genes, segment S, genomic RNA, isolate 38K 99 CHUV F Chuzan virus genomic RNA for non structural protein NS1, complete cds 98 R Chuzan virus genomic RNA for non structural protein NS1, complete cds 95 IBAV F Epizootic hemorrhagic disease virus (serotype 2 / strain Ibaraki) segment 3 92 R Epizootic hemorrhagic disease virus (serotype 2 / strain Ibaraki) segment 3 99 BEFV F Bovine ephemeral fever virus isolate CS1935 surface glycoprotein (G) gene, partial cds 99 R Bovine ephemeral fever virus isolate CS1931 surface glycoprotein (G) gene, partial cds 99

F, Forward; R, Reverse. Supplementary Fig. 1

Wanju Jinan

Gimje

Namwon Gochang

Jeollabuk-do, Korea

Supplementary Fig S1. Five cattle farms where collected Culi- coides biting midges in Jeollabuk-do, Korea. Culicoides biting midges were collected from 5 different cattle farms located in Gimje, Gochang, Wanju, Jinan, and Namwon areas, Jeollabuk- do. Two farms (Gimje and Gochang) were located in plain area with low altitude (about 50 m), and 3 farms (Wanju, Jinan, and Namwon) were mountainous areas with relatively high altitude (above 300 m).